Introduction:
Maternally-inherited endosymbionts infecting arthropods are one of the
most diverse and abundant of all bacteria infecting them. About
two-third of terrestrial arthropods are infected with at least one
maternally-inherited endosymbiont (Hilgenboecker, Hammerstein,
Schlattmann, Telschow, & Werren, 2008; Weinert, Araujo-Jnr, Ahmed, &
Welch, 2015). These endosymbionts play crucial role in the ecology and
evolution of their hosts (Gebiola et al., 2017; Semiatizki et al.,
2020). The most abundant of these are Wolbachia, Cardinium,
Arsenophonus, Rickettsia and Spiroplama. Out of these,Wolbachia remains the most widely distributed endosymbiont with
an incidence rates of 16-66% (Hilgenboecker et al., 2008; Werren,
Windsor, & Guo, 1995) and 18 different clades (supergroup A to R)
reported in different hosts across the world (Landmann, 2019). Incidence
of the other endosymbionts varies from 4-10% (Duron et al., 2008;
Zchori-Fein & Perlman, 2004).
The key factor explaining this abundance has been their ability to jump
from one host to the other, in spite of being vertically transmitted
from mother to offspring (Werren, Baldo, & Clark, 2008). As a result,
these endosymbionts rarely show congruence with the host phylogeny,
indicating substantial horizontal transfer across evolutionary
timescales to taxonomically unrelated hosts (Shoemaker et al., 2002;
Werren, Zhang, & Guo, 1995). This is also evident from the occurrence
of similar endosymbiont strains in taxonomically unrelated hosts and
conversely, the presence of divergent strains in closely related hosts
(Vavre, Fleury, Lepetit, Fouillet, & Bouletreau, 1999). Individual
arthropods can harbor multiple strains of one endosymbiont as well as
multiple strains of different endosymbionts (Zélé et al., 2018) which
perhaps indicates that different endosymbionts can use the same host to
spread across different arthropod communities.
Another key feature of endosymbionts is the pervasive recombination seen
in their genomes (Ellegaard, Klasson, Naslund, Bourtzis, & Andersson,
2013). This has been particularly well documented in Wolbachia(Malloch & Fenton, 2005) as well as other endosymbionts (Mouton et al.,
2012). The level of recombination is so extensive that single gene
sequences are unable to properly reflect the evolutionary history of a
strain. Unsurprisingly, this has necessitated the development of multi
locus strain typing (MLST) system (Maiden et al., 1998). The results of
such MLST surveys revealed the extent of recombination to be similar to
those of pathogenic free-living bacteria (Yahara et al., 2016). This is
surprising because, unlike free-living bacteria, most endosymbionts
cannot survive outside the host. Therefore, this extensive recombination
must be an outcome of the horizontal transfer of strains across
arthropod communities, since, for recombination to happen, two
endosymbionts must come in contact within one host cytoplasm. As new
strains are horizontally transferred to novel hosts; they encounter
resident endosymbionts and thereby increase opportunities of
recombination between them. Evidence for such recombination is also well
documented. The parasitoid wasp Nasonia and its hostProtocalliphora show the presence of a very similar recombinantWolbachia (Werren & Bartos, 2001). Recombinant Wolbachiastrains have also been reported in Anastrepha fruit flies and
their parasitoid braconid wasps (Mascarenhas, Prezotto, Perondini,
Marino, & Selivon, 2016).
Horizontal transfer, therefore, can explain at least two major
characteristics of endosymbionts, their wide distribution as well as the
recombinant nature of their genomes. A major question that emerge from
this is, at what level of biological organization are these horizontal
transfers taking place? A relatively simple way to uncover this level is
to enumerate specific ecological interactions where close associations
between the two interacting arthropods have been implicated in
horizontal transfer. These include host-parasite, host-parasitoid,
prey-predator and other ecological relationships. Examples where
host-parasitoid interactions have been implicated for such transfer
include the presence of similar Wolbachia strains among
frugivorous Drosophila and their hymenopteran parasitoid (Vavre
et al., 1999), Nasonia vitripennis and Muscidifuraxuniraptor sharing similar Wolbachia with their fly hostProtocalliphora (Baudry, Bartos, Emerson, Whitworth, & Werren,
2003), transmission of Wolbachia into whitefly via parasitoid
wasps (Ahmed, Breinholt, & Kawahara, 2016). Another such ecological
association which can lead to endosymbiont transfer is prey-predator
relationships like the predatory mite Metaseiulus occidentalisand its prey Tetranychus urticae (spider mite) sharing similar
endosymbionts (Hoy & Jeyaprakash, 2005). Parasites like mites can also
facilitate the transfer of Wolbachia to Drosophila host
populations (Brown & Lloyd, 2015). These can also be host plant
mediated transfer of Cardinium to different leaf hopper species
(Gonella et al., 2015) as well as horizontal transfer ofWolbachia in whitefly via cotton leaves (Li et al., 2017).
It is clear from these examples that these horizontal transfers are
taking place when two hosts are coming together to perform a particular
ecological function. The endosymbiont present within these hosts are
then serendipitously getting transferred from one host to the other.
Therefore, to understand the dynamics of the spread of endosymbionts
through horizontal transfer, one needs to look at the level where most
of these ecological associations are taking place, which is within a
particular ecological community. A well-defined ecological community
will have several diverse host taxa interacting with each other,
thereby, facilitating horizontal transfer. Moreover, many host taxa can
belong to many different ecological communities (Morrow, Frommer,
Shearman, & Riegler, 2014). This cosmopolitan nature of a few host taxa
will further facilitate the spread of endosymbionts from one ecological
community to another, almost like spreading through a metacommunity
(Brown, Mihaljevic, Des Marteaux, & Hrček, 2020). Therefore,
investigating endosymbiont diversity and horizontal transfer within
specific ecological communities seems logical. Yet, there are very few
studies that have taken this approach and instead focus mainly on
endosymbiont spread within a particular habitat (Stahlhut et al., 2010),
or in a specific genus (Baldo et al., 2008; Raychoudhury, Baldo,
Oliveira, & Werren, 2009) or within specific taxa (Ahmed et al., 2016).
Amongst community-wide surveys, Kittayapong, Jamnongluk, Thipaksorn,
Milne, and Sindhusake (2003), demonstrated Wolbachia strain
diversity within rice field arthropod community. Sintupachee, Milne,
Poonchaisri, Baimai, and Kittayapong (2006), reported plant-mediated
horizontal transfer among arthropod community found on pumpkin leaves.
Most of these studies are based on single gene phylogenies which makes
identification of recombination difficult. An important corollary of
this view of within-community horizontal transfer of endosymbionts can
lead to another important hypothesis about sequence diversity of the
endosymbionts themselves. If endosymbionts are rapidly undergoing
horizontal transfer within a particular ecological community then very
similar bacterial strains would be found among the arthropod hosts of
that community. This would make these bacteria more closely related to
each other, than expected, resulting in lower than expected pairwise
sequence divergence among them. This lower than expected levels of
sequence divergence can serve as a signature of recent and relatively
rapid community-wide horizontal transfer of resident endosymbionts.
In the present study, we try to answer whether such relatively rapid
horizontal transfer and resulting recombination are happening within the
endosymbionts of a diverse soil arthropod community. Three major
endosymbionts, Wolbachia, Cardinium and Arsenophonus , were
selected and screened across arthropod hosts. We investigatedWolbachia sequence diversity using the well-established MLST
scheme (Baldo et al., 2006) and also identified specific recombination
events. We also investigated Cardinium and Arsenophonusincidence but with single gene sequences. A statistical model was then
used to test whether the endosymbiont found within this community are
more closely related to each other than expected.